KR101672138B1 - Composition comprising Cudraflavanone D or Steppogenin isolated from Cudrania tricuspidata as a standard for the preventing and treating of inflammatory diseases - Google Patents

Composition comprising Cudraflavanone D or Steppogenin isolated from Cudrania tricuspidata as a standard for the preventing and treating of inflammatory diseases Download PDF

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KR101672138B1
KR101672138B1 KR1020150106965A KR20150106965A KR101672138B1 KR 101672138 B1 KR101672138 B1 KR 101672138B1 KR 1020150106965 A KR1020150106965 A KR 1020150106965A KR 20150106965 A KR20150106965 A KR 20150106965A KR 101672138 B1 KR101672138 B1 KR 101672138B1
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disease
steppogenin
cudraflavanone
inflammatory
cells
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KR1020150106965A
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Korean (ko)
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김윤철
오현철
고원민
김동철
이동성
윤치수
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원광대학교산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/60—Moraceae (Mulberry family), e.g. breadfruit or fig
    • A61K36/605—Morus (mulberry)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system

Abstract

The Cudraflavanone D and steppogenin of the present invention are compounds isolated from cucurbitaceous trees. In the present invention, it was confirmed that the coadaplavanone D and stepojenin were excellent in the effect of inhibiting inflammation in mouse-derived microglia, BV2 cells, and thus the inhibitory effect of inflammatory mediators was also apparent. The anti-inflammatory effects of the copla-laparavone D and stepojenin are known to be related to the nuclear factor kappa-light-chain-enhancer of activated B (NF-kB) and mitogen- activated protein kinase pathway. Cudrabravanone D and stepogensin inhibited the overexpression of NF-kB and MAPK pathway, and thus showed excellent anti-inflammatory effects.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing and treating inflammatory diseases, which comprises as an active ingredient coudraplavanone D or stepogenin, }

The present invention relates to a composition for treating and preventing an inflammatory disease, which comprises a compound isolated from Curdrania tricuspidata as an active ingredient, and more particularly, to a composition for treating or preventing inflammatory diseases comprising Cudraflavanone D or Cudraflavanone D The present invention relates to a pharmaceutical composition and a health functional food for the prevention and treatment of microglia cell-associated inflammatory diseases containing steppogenin as an active ingredient.

Inflammation is caused by harmful substances or damage by chemical stimulation before the body's defense against external stimuli. When an inflammatory reaction occurs, inflammatory mediators are created, leading to symptoms such as fever, fever, swelling, pain, and dysfunction. In addition to activating the oxidative stress transcription factor nuclear factor kappa-b (NF-κB), reactive oxygen species generated from the inflammatory reaction are also important signal transducer inducible nitric oxide synthase (iNOS) and inflammatory inducer cyclooxygenase (COX-2), thereby promoting the inflammatory response, leading to abnormal mutations and cancer.

Microglial cells play a central role in the inflammatory response in the central nervous system involved in various neuropathic diseases. In the present invention, a molecular microphysiological study on the inflammatory response induced by LPS, a compound isolated from cucurbitan tree, Cudraflavanone D or steppogenin, was studied using a microglial cell model .

Curdrania tricuspidata is a deciduous broad-leaved arboreous tree belonging to the genus Molaceae. It is distributed mainly in East Asia including Korea, and 10 species are known. In Korea, only one species is native, and morin papulinin ), Stachydrine, kaempferol-7-glucoside, and the like.

However, no studies have been reported on the effects of Cudraflavanone D and Steppogenin on microglial cells associated with inflammatory diseases in the literature.

In this invention, Cudraflavanone D and Steppogenin isolated from Cucumber japonica have excellent effect of inhibiting inflammation in mouse-derived microglia, BV2 cells, and thus the inflammatory mediator The inhibitory effect was also evident. The anti-inflammatory effects of the copla-laparavone D and stepojenin are known to be related to the nuclear factor kappa-light-chain-enhancer of activated B (NF-kB) and mitogen- activated protein kinase pathway. Cudrabravanone D and stepogenin inhibit the activity of overexpressed NF-kB and MAPK pathway, confirming that the anti-inflammatory effect is excellent, and thus a composition and a composition for prevention and treatment of microglial cell- And thus the present invention has been completed.

Related prior arts are Korean Patent No. 10-0701607 entitled " Composition for prevention and treatment of cancer containing quadruplavanone A separated from cucumber tree having inhibitory activity of topoisomerase I "and Korean Patent No. 10 -1355821 "A composition for the prevention and treatment of alcoholic gastrointestinal tract diseases containing an extract of Cedarwood bean as an active ingredient ".

The present invention provides a pharmaceutical composition and a health functional food for the prevention and treatment of anti-inflammatory, which comprises as an active ingredient coudraplavanone D or stepogensin, which is a compound isolated from cheddar.

The present invention provides a pharmaceutical composition for the prevention and treatment of anti-inflammatory activity comprising Cudraflavanone D or Steppogenin compound isolated from Curdrania tricuspidata as an active ingredient.

The compound is obtained by extracting Curdrania tricuspidata with methanol; Dissolving the methanol extract in water and obtaining a chloroform fraction using a solution-solution dispensing method with chloroform; And separating the chloroform fraction through fractionation using silica gel column chromatography.

The anti-inflammatory agent is characterized in that it is at least one selected from the group consisting of Lou Gehrig's disease, Parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis, multiple nervous atrophy, epilepsy, encephalopathy and stroke.

The inflammation-inhibiting pathway of the Cudraflavanone D or Steppogenin compound is characterized by inhibiting the p-JNK or p-38 pathway.

In addition, the present invention provides a health functional food for preventing and improving anti-inflammation comprising Cudraflavanone D or Steppogenin compound isolated from Curdrania tricuspidata as an active ingredient.

The present invention relates to an anti-inflammatory effect which comprises quadruplavanone D or stepogenin, which is isolated from cucurbitaceous tree, as an active ingredient. Cucurbitan is a natural resource and has excellent cytotoxicity, The compounds of the present invention are useful as pharmaceutical compositions and health functional foods for the prevention and treatment of various inflammatory diseases by inhibiting the pathway of factors important for inflammatory reaction and having an anti-inflammatory effect.

FIG. 1 is a view showing a method of separating codaplavanone D or stepojenin from Coleoptera.
FIG. 2 is a diagram showing the structure of coudraplavanone D, showing the structure identification (A) and 13 C-NMR (B) by 1 H-NMR and the structure identification (C).
FIG. 3 is a graph showing the cell survival rate for evaluating cytotoxicity of cupraflavanone D. FIG.
FIG. 4 is a graph showing the inhibitory effect of TNF-α (A), IL-1β (B), IL-12 (C) and IL-6 (D) mRNAs on the inflammatory cytokines of coadaplavanone D.
5 is a graph showing the effect of suppressing (C) the NO production inhibitory effect (A), the PGE 2 generation inhibitory effect (B), and the iNOS and COX-2 protein expression levels of cupraflavavanone D.
Figure 6 shows the effect (A) of inhibiting IκB-α degradation and phosphorylated IκB-α in the cytoplasmic fraction of codraprapavanone D, the transcriptional repression inhibition of NF-kB p65 and p50 in the cytoplasmic fraction (B) (E) suppresses the transcriptional repression inhibitory effect of p65 and p50 (C), the transcriptional repression inhibitory effect of p50 on the nucleus by immunofluorescence staining (D), and the binding activity of NF-kB in the nuclear fraction Fig.
7 is a graph showing an inhibitory effect on the mitogen activated protein kinase (MAPKs) activity pathway ERK (A), JNK (B), and p38 (C) of codaplavanone D. FIG.
FIG. 8 is a diagram showing the structure of steppenine. FIG. 12A shows structure identification by 1 H-NMR, FIG. 13B shows structure identification by 13 C-NMR, and FIG.
FIG. 9 is a graph showing cell viability to examine cytotoxicity of stepojenin. FIG.
FIG. 10 is a graph showing the inhibitory effect of steppenine on inflammatory cytokines TNF-α (A), IL-1β (B), IL-12 (C) and IL-6 (D) mRNA.
Fig. 11 is a graph showing the effect of inhibiting NO production (step A) of stephenin, the effect of inhibiting PGE 2 production, and the inhibitory effect (C) of iNOS and COX-2 protein expression.
Figure 12 shows the effect of inhibiting IκB-α degradation and phosphorylated IκB-α in the cytoplasmic fraction of stepogenin (A), the inhibition of transcription of NF-kB p65 and p50 in the cytoplasmic fraction (B) (E) suppression of the transcriptional repression inhibitory effect (C) of p50, the suppression effect of transfer of p50 into the nucleus (D) by immunofluorescence staining, and the binding activity of NF-kB in nuclear fraction to be.
FIG. 13 is a graph showing inhibitory effects of steppenine on mitogen activated protein kinase (MAPKs) activation pathways ERK (A), JNK (B) and p38 (C).

The present invention provides a pharmaceutical composition for the prevention and treatment of anti-inflammation comprising Cudraflavanone D or Steppogenin as an active ingredient.

The Cudraflavanone D or Steppogenin of the present invention is characterized in that it is isolated from Curdrania tricuspidata, which is a deciduous broad-leaved tree of Morus alba.

The anti-inflammation defined in the present invention collectively refers to anti-inflammation occurring in microglial cells, and includes but is not limited to anti-inflammatory related to neuropathic diseases, preferably Lou SS, Parkinson's disease, Huntington's disease, Alzheimer's disease, Multiple sclerosis, multiple nervous atrophy, epilepsy, encephalopathy, and stroke.

The compounds of the present invention may be prepared into pharmaceutically acceptable salts and solvates by methods conventional in the art.

Pharmaceutically acceptable salts include acid addition salts formed by free acids. The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration.

As the free acid, organic acids and inorganic acids can be used. As the inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acids include methanesulfonic acid, p- toluenesulfonic acid, acetic acid, trifluoroacetic acid, Maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycollic acid, gluconic acid glutamic acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, and the like can be used.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt in particular, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the compounds of the present invention include, unless otherwise indicated, salts of acidic or basic groups that may be present in the compounds of the present invention. For example, pharmaceutically acceptable salts include the sodium, calcium and potassium salts of the hydroxy groups

Other pharmaceutically acceptable salts of amino groups include hydrobromide, sulphate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (Mesylate) and p-toluenesulfonate (tosylate) salts, which can be prepared by methods known to those skilled in the art or by the preparation of salts.

The present compounds of the present invention can be used for the treatment of lipopolysaccharide (LPS) -induced microglial cell damage. NF-kB (nuclear factor kappa-light-chain-enhancer of activated B) and mitogen-activated protein kinase (MAPK) pathway, thereby confirming that it can be effectively used as a pharmaceutical composition and a health functional food for the prevention and treatment of anti-inflammation.

The pharmaceutical composition for preventing and treating anti-inflammation of the present invention comprises 0.1 to 50% by weight of the above compound based on the total weight of the composition.

However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

Since the compound itself of the present invention has little toxicity and side effects, it can be safely used for long-term use for preventive purposes.

The pharmaceutical compositions comprising the compounds of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions, Examples of carriers, excipients and diluents that can be included in the composition including the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. Various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. may be included in addition to water and liquid paraffin which are simple diluents used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the composition of the present invention can be determined in consideration of the condition and body weight of the patient, the degree of disease, the type of drug, the route of administration, and the absorbance, inactivity and the drug to be used in combination with the active ingredient in the body, (Body weight) to 500 mg / kg (body weight), 0.1 mg / kg (body weight) to 400 mg / kg (body weight) or 1 mg / kg (body weight) to 300 mg / kg (body weight), and may be administered once or several times in divided doses. The dose is not intended to limit the scope of the invention in any way. And may be suitably selected by those skilled in the art.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In addition, the present invention provides a health functional food for preventing and improving anti-inflammation comprising a compound isolated from a tree as an active ingredient.

In addition, the present invention provides a food or food additive containing, as an active ingredient, a compound isolated from the above-mentioned Cucumber tree having the effect of preventing and improving neurological diseases.

The composition comprising the compound of the present invention can be used variously for medicines, foods and drinks for the prevention and improvement of neurological diseases.

Examples of foods to which the compound of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, health supplements and the like, and they can be used in powder, granule, tablet, have.

The compounds of the present invention can be added to foods or beverages for the purpose of preventing and improving neurological diseases. At this time, the amount of the compound in the food or beverage may generally be from 0.01 to 15% by weight of the total food weight of the health food composition of the present invention, and the health beverage composition is preferably 0.02 to 10 g based on 100 ml, Can be added at a ratio of 0.3 to 1 g.

The health beverage composition of the present invention contains the above-mentioned compound as an essential ingredient in the indicated ratio, and there is no particular limitation on the liquid ingredient, and the ordinary beverage can contain various flavors or natural carbohydrates as an additional ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose and sucrose and polysaccharides such as dextrin and sludge dextrin Conventional sugars and sugar alcohols such as xylitol, sorbitol, and erythritol. (Taurine, or stevia extract (for example, glycyrrhizin containing rebaudioside A) and synthetic flavors (saccharin, aspartame, etc.) as flavors other than the above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 mL of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like.

In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention

Hereinafter, the present invention will be described in detail with reference to the following experimental examples.

However, the following experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following experimental examples.

Example  One. From the cedarwood tree,  D ( Cudraflavanone  D)

A method for separating Cudraflavanone D is shown in FIG. As shown in FIG. 1, 300 g of a 100% methanol extract was obtained by allowing the cucumber (6 kg) to stand at room temperature for 24 hours in 100% methanol. The 100% methanol extract was dissolved in water, and 160 g of a chloroform fraction was obtained using a solution-solution partitioning method using chloroform. The resulting chloroform fractions were fractionated using silica gel CC column chromatography (20%, 40%, 60%, 80%, 100% (v / v) EtOAc in hexane) . Four fractions (45 g) were obtained by silica gel CC column chromatography using chloroform: ethyl acetate = 10: 1 ratio. Four fractions were separated by silica gel CC column chromatography using hexane: acetone = 5: 1, 4: 1, 3: 1, 2: . The second fraction thereof was subjected to RP 18 column chromatography to obtain 60 mg of coudraplavanone D using a methanol: water = 3: 1 ratio solvent.

In order to identify the structure of the obtained coudralavanone D, the structure was identified using 1 H-NMR of FIG. 2A and 13 C-NMR of FIG. 2B, and the results as shown in FIG. 2C were obtained.

brown oil. 1 H-NMR (MeOD, 400 MHz) δ 5.58 (dd, J = 3.2, 12.4 Hz, H-2), 3.03 (dd, J = 12.0, 17.2 Hz, H-3a), 2.71 (dd, J = 3.2 , 17.2 Hz, H-3b) , 5.93 (s, H-8), 6.32 (s, H-3 '), 7.03 (s, H-6'), 3.18 (d, J = 7.2 Hz, H-11 ), 5.18 (m, H- 12), 1.73 (s, H3-14), 1.65 (s, H3-15), 3.18 (d, J = 7.2 Hz, H-16), 5.25 (m, H-17 ), 1.63 (s, H3-19), 1.69 (s, H3-20). 13 C-NMR (MeOD, 100 MHz) δ 75.9 (C-2), 43.1 (C-3), 198.4 (C-4), 162.4 (C-5), 109.4 (C-6), 165.8 (C- (C-10), 117.5 (C-1 '), 154.3 (C-2'), 103.3 (C-3 '), 156.8 C-4 '), 120.5 (C-5'), 128.6 (C-6 '), 21.8 (C-11), 123.9 25.9 (C-15), 28.6 (C-16), 124.3 (C-17), 132.5 (C-18), 25.9 (C-19), 17.7 (C-20).

Experimental Example  1. Mouse-derived microglia, BV2 cell culture and cytotoxicity

Mouse microsomal BV2 cells (2 × 10 5 cells / well) were mixed with 10% heat-inactivated FBS, penicillin G (100 IU / ml, Gibco, 15240062) and streptomycin (100 μg / Gibco, 15240062), cultured in a 5% carbon dioxide incubator (Sanyo, MCO175) at 37 ° for 24 hours, treated with the concentration of codraprapavanone D for 48 hours, and cultured in a 5% CO 2 incubator And cell viability was measured using MTT method. In addition, all the experimental values were expressed as the average cell survival rate for the control group, and were calculated using the experiment values repeated three times.

In order to measure cytotoxicity, primary cultured macrophages (1.25, 2.5, 5, 10 μM), which was isolated from abdominal cavity of mice, were treated for 48 hours and cell viability was measured. As a result, D, the cell survival rate remained high regardless of the concentration (Fig. 3).

Experimental Example  2. Mouse-derived microglia BV2 cells  Inhibitory effect of inflammatory cytokines TNF-α, IL-1β, IL-12, and IL-6 on mRNA expression

BV2 cells, which are mouse-derived microglia, were treated with 1.25, 2.5, 5, and 10 μM of Cdraplavanone D for 3 hours, followed by induction of inflammatory reaction with lipopolysaccharide (LPS) % CO2 incubator. As a result of the RT-PCR, TNF-α (FIG. 4A), IL-1β (FIG. 4B), IL-12 (FIG. 4C) 6 mRNA expression (Fig. 4D) was inhibited in a concentration-dependent manner when treated with 1.25, 2.5, 5, or 10 μM of codraprapavanone D.

Experimental Example  3. In BV2 cells, a mouse-derived microglia, inflammation mediators NO, PGE 2 , inhibitory effect of iNOS and COX-2 protein expression

To investigate the production of NO, PGE 2 and the inhibition of protein expression of iNOS and COX-2, which are inflammatory mediators in BV2 cells of cupraclavanone D, Kim et al., European Journal of Pharmacology, 721, pp. 267-276, 2013).

BV2 cells were pretreated with 1.25, 2.5, 5 and 10 μM of codraplavanone D for 3 hours, and then induced with inflammatory reaction with lipopolysaccharide (LPS) and cultured in a 5% CO 2 incubator for 24 hours. As shown in FIG. 5A and FIG. 5B, NO and PGE 2 production, which were increased when lipopolysaccharide (LPS) was treated, were found to be higher when the concentration of Cdraplavanone D was 1.25, 2.5, Dependent inhibition of the inhibitory effect.

Specifically, the western blot analysis was carried out in order to confirm the effect of the above-mentioned codraplavanone D and the treatment time to promote iNOS and COX-2 protein expression.

First, BV2 cells were cultured for 12 hours at a concentration of 3 × 10 6 cells / ml in a 60 mm dish, treated with lupopolysaccharide at a concentration of 1 μg / ml, treated with coudraplavanone D for 24 hours, . After the above cultivation, RIPA (89900, Thermo) buffer was added and the supernatant was separated by centrifugation at 4 DEG C and 14,000 X g for 25 minutes. Protein quantification in the supernatant was performed using a BSA protein kit (Pierce Biotechnology).

Specifically, the supernatant was subjected to electrophoresis for 2 hours using 7.5% SDS-polyacrylamide gel, and then transferred from the polyacrylamide gel using a nitrocellulose membrane (NC membrane) Respectively. The transferred nitrocellulose membrane was terminated in a blocking buffer (0.1% Tween 20 in Tris-buffered saline) containing 5% nonfat milk for 1 hour. After the reaction was stopped, the membrane was washed with PBST (PBS, 0.1% Tween 20) three times, once every 10 minutes, and then iNOS (SC-650, Santacruz biotechnology, USA), COX-2 (SC- Biotechnology, USA) was diluted 1: 1000 and reacted for 1 hour. After the reaction, the cells were washed three times with the PBST once every 10 minutes, and the secondary antibody (anti-rabbit IgG, SC-2004, Santacruz biotechnology, USA) was diluted 1: 1000 and reacted for 1 hour.

After the reaction, the plate was washed three times with PBST once every 10 minutes, and then mixed with ECL (Amersham Pharmacia Biotech) solution at a ratio of 1: 1 and poured on a nitrocellulose membrane (NC membrane) to emit light. The film was sensitized and developed. Actin content was measured in the same manner using an antibody against actin (SC-1616, Santacruz biotechnology, USA).

As shown in FIG. 5C, it was confirmed that the protein expression of iNOS and COX-2, which are known as inflammatory mediators, is significantly suppressed as the concentration of the copla laparavone D increases.

Experimental Example  4. Mouse-derived microglia in BV2 cells IκBα  Decomposition inhibition, Iκ Inhibition of Bα phosphorylation, inhibition of NFκB (p65, p50) nuclear transfer and inhibition of NFκB p65 binding ability

We examined the effect of coadaplavanone D on the NFκB-related pathway in BV2 cells. To investigate inhibition of IκBα degradation, inhibition of IκBα phosphorylation, inhibition of NFκB p65 and p50 nuclear transfer, and inhibition of NFκB p65 binding ability, the method of the above-mentioned method (Kim et al., European Journal of Pharmacology, 721, pp. 267-276, The experiment was carried out using western blot analysis, which is the same method as in Experimental Example 3.

BV2 cells were treated with 2.5, 5, and 10 μM of Cudrablavanone D for 3 hours, followed by induction of the inflammatory response pathway with lipopolysaccharide, followed by incubation in a 5% CO 2 incubator for 1 hour. SC-7178, Santacruz biotechnology, USA) inhibition of IκBα (SC-371, Santacruz biotechnology, USA) degradation, IκBα phosphorylation (p-IκBα, SC-8404, Santacruz biotechnology, USA) USA) Nuclear separation experiments were carried out to confirm nuclear transfer inhibition and NFκB p65 binding ability. 6A and 6B show results in the cytoplasm, and FIGS. 6C and 6E show experimental results in the nucleus. As shown, IκBα degradation and IκBα phosphorylation (FIG. 6A), Nuclear transfer of NFκB p65 and p50 and NFκB p65 binding capacity (FIG. 6B, FIG. 6C), which were increased when lipopolysaccharide was treated, 5, and 10 μM, respectively. FIG. 6D is an experiment using immunofluorescence staining, and FIG. 6E is a graph thereof. Experiments were carried out using the method of Kim et al., European Journal of Pharmacology, 721, pp. 267-276, 2013. As a result of the experiment, it was confirmed that NFκB p50 transfection into nucleus when treated with lipopolysaccharide was inhibited by treatment with 10 μM of codraplavanone D.

Experimental Example  In BV2 cells, a mouse-derived microglia, Mito Inhibitory effect of gene active protein kinase expression

Suppression of p-ERK, p-JNK, and p-38 expression, which are mitogen-activated protein kinase (MAPK) species, against inflammatory response induced by lipopolysaccharide in BV2 cells of Kudraplavanone D Using the method of Kim et al., European Journal of Pharmacology, 721, pp. 267-276, 2013 and Western blot analysis, which is the same method as in Experimental Example 3, Respectively.

BV2 cells were pretreated with 2.5, 5, 10 μM of Cudrablavanone D for 3 hours, and then cultured in a 5% CO 2 incubator for 1 hour with induction of inflammatory response pathway by lipopolysaccharide. As a result, the expression of p-ERK (Fig. 7A), p-JNK (Fig. 7B) and p-38 (Fig. 7C) was detected by lipopolysaccharide Respectively. However, the expression of p-JNK (Fig. 7B) and p-38 (Fig. 7C) was gradually decreased and the expression of p-ERK (Fig. 7A) was not changed. This indicates that the inflammation-inhibiting pathway of the copla-laparavone D is p-ERK, p-JNK, which is a type of mitogen activated protein kinase (MAPKs), p-JNK ethyl acetate = 10: 1 ratio solvent was used to obtain four fractions. Four fractions were separated by silica gel CC column chromatography using hexane: acetone = 5: 1, 4: 1, 3: 1, 2: . The third fraction was purified by silica gel CC column chromatography to obtain 65 mg of stepogensin using hexane: ethyl acetate = 3: 1 ratio solvent.

In order to identify the structure of the obtained steppogenin, 1 H-NMR of FIG. 8A and 13 C-NMR of FIG. 8B were used to identify the structure, and the results of FIG. 8C were obtained.

, p-38 pathway.

Example 2. Isolation of Steppogenin from Cucurbitaceae

A method of isolating steppogenin is shown in Fig. As shown in FIG. 1, 300 g of a 100% methanol extract was obtained by allowing the cucumber (6 kg) to stand at room temperature for 24 hours in 100% methanol. The 100% methanol extract was dissolved in water, and 160 g of a chloroform fraction was obtained using a solution-solution partitioning method using chloroform. The resulting chloroform fractions were fractionated using silica gel CC column chromatography (20%, 40%, 60%, 80%, 100% (v / v) EtOAc in hexane) . Fourth fraction (45 g) was purified by column chromatography on silica gel (C.

brown solid. 1 H-NMR (DMSO- d6, 400 MHz) δ 5.61 (dd, J = 2.8, 13.2 Hz, H-2), 3.25 (dd, J = 13.2, 17.2 Hz, H-3a), 2.62 (dd, J = 2.8, 17.2 Hz, H- 3b), 5.89 (br s, H-6, H-8), 6.37 (d, J = 1.6 Hz, H-3 '), 6.29 (d, J = 1.6, 8.4 Hz , H-5 '), 7.20 (d, J = 8.4 Hz, H-6'). 13 C-NMR (DMSO- d6, 100 MHz) δ 74.0 (C-2), 41.3 (C-3), 197.0 (C-4), 163.7 (C-5), 95.9 (C-6), 166.7 ( C-7), 95.1 (C-8), 163.6 (C-9), 101.9 (C-10), 115.6 158.8 (C-4 '), 106.7 (C-5'), 128.4 (C-6 ').

Experimental Example  6. Mouse-derived microglia, BV2 cell culture and cytotoxicity

Mouse microsomal BV2 cells (2 × 10 5 cells / well) were mixed with 10% heat-inactivated FBS, penicillin G (100 IU / ml, Gibco, 15240062) and streptomycin (100 μg / Gibco, 15240062), cultured in a 5% CO 2 incubator at 37 ° for 24 hours, treated with steppogenin in a concentration-dependent manner, cultured in a 5% CO 2 incubator for 48 hours, Cell viability was measured by MTT assay. In addition, all the experimental values were expressed as the average cell survival rate for the control group, and were calculated using the experiment values repeated three times.

In order to measure cytotoxicity, mouse microglia BV2 cells were treated with 10, 20, 40, 80 μM steppogenin for 48 hours and cell viability was measured. When the steppogenin was treated at a concentration of 80 μM or less, The cell survival rate remained high regardless of the concentration of the genin (FIG. 9).

Experimental Example  7. Mouse-derived microglia BV2 In a cell  Inhibitory effect of TNF-α, IL-1β, IL-12, and IL-6 mRNA on inflammatory cytokines

BV2 cells, a mouse-derived microglia, were treated with steppenine 10, 20, 40, and 80 μM for 3 hours, pretreated with lipopolysaccharide (LPS) 2 incubator. As a result of the RT-PCR, TNF-α (FIG. 10A), IL-1β (FIG. 10B), IL-12 (FIG. 10C) and IL-6 10D) mRNA expression was inhibited by 10, 20, 40, and 80 μM of steppenine in a concentration-dependent manner.

Experimental Example  8. In BV2 cells, a mouse-derived microglia, inflammation mediators NO, PGE 2 , And iNOS and COX-2 protein expression inhibitory effect

In order to examine inhibition of protein expression of NO, PGE 2 , iNOS and COX- 2 , which are inflammatory mediators in BV2 cells of steppenin, Kim et al., European Journal of Pharmacology, 721, pp. 267-276, 2013) and western blot analysis (same method as Experimental Example 3).

As shown in FIG. 10, the increase in NO (FIG. 11A), the increase in PGE 2 (Fig. 11B), the effect of the step-dependent inhibition of steppenanin at 10, 20, 40, and 80 μM was confirmed. As a result, as shown in FIG. 11C, it was confirmed that the expression of iNOS and COX-2 protein, which are known as inflammatory mediators, is significantly suppressed as the concentration of stepogenin increases.

Experimental Example  9. Mouse-derived microglia in BV2 cells IκBα  Decomposition inhibition, Iκ Bα phosphorylation inhibition, NFκB  (p65, p50) Nucleus  In addition, NFκB  p65 Cohesion  Inhibitory effect

The effects of stepojenin on NFκB-related pathways in BV2 cells were examined. Methods for the inhibition of IκBα decomposition, inhibition of IκBα phosphorylation, inhibition of NFκB p65 and p50 nuclear transfer, and inhibition of NFκB p65 binding ability were carried out by the method of Kim et al., European Journal of Pharmacology, 721, pp. 267-276, The experiment was carried out using western blot analysis, which is the same method as in Experimental Example 3.

BV2 cells were pretreated with 20, 40, and 80 μM stepepogenin for 3 hrs. After induction of the inflammatory response pathway with lipopolysaccharide, cells were cultured in a 5% CO 2 incubator for 1 hr. SC-7178, Santacruz biotechnology, USA) inhibition of IκBα (SC-371, Santacruz biotechnology, USA) degradation, IκBα phosphorylation (p-IκBα, SC-8404, Santacruz biotechnology, USA) USA) nuclear transfer inhibition and NFκB p65 binding ability. 12A and 12B show results in the cytoplasm, and FIGS. 12C and 12E show experimental results in the nucleus. As shown, IκBα degradation and IκBα phosphorylation (FIG. 12A), NFκB p65 and p50 nuclear transfer and NFκB p65 binding ability (FIG. 12B, FIG. 12C) increased when treated with lipopolysaccharide (LPS) , 40, and 80 μM, respectively. FIG. 12D is an experiment using immunofluorescence staining, and FIG. 12E is a graph thereof. Experiments were carried out using the method of Kim et al., European Journal of Pharmacology, 721, pp. 267-276, 2013. As a result of the experiment, it was confirmed that transcription of NFκB p50 into nucleus when treated with lipopolysaccharide was suppressed when steppinginin was treated at 80 μM.

Experimental Example  10. In mouse-derived microglia, BV2 cells, beauty Inhibitory effect of tolan-activated protein kinase expression

Expression of p-ERK, p-JNK, and p-38, which are mitogen-activated protein kinase (MAPKs), on lipopolysaccharide (LPS) -induced inflammatory response in BV2 cells of stepheninine In order to identify the inhibitory pathway, the method of Kim et al., European Journal of Pharmacology, 721, pp. 267-276, 2013 and Western blot analysis, which is the same method as in Experimental Example 3, Respectively.

BV2 cells were pretreated with 20, 40, and 80 μM of steppenine for 3 hours, and induced with inflammatory response by lipopolysaccharide, followed by culture in a 5% CO 2 incubator for 1 hour. 13 (A), p-JNK (FIG. 13B), and p-38 (FIG. 13C) expression by lipopolysaccharide (LPS) Respectively. However, when stepheninin was treated at different concentrations, the expression of p-JNK (Fig. 13B) and p-38 (Fig. 13C) gradually decreased and the expression of p-ERK (Fig. 13A) remained unchanged. This suggests that the pathogenesis of stepinin inhibition is inhibited by p-ERK, p-JNK, p-JNK, and p-38 pathways, which are mitogen-activated protein kinases (MAPKs) .

Claims (5)

Parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis, multiple nervous atrophy (Cushing's syndrome), including Cudraflavanone D or Steppogenin compounds isolated from Curdrania tricuspidata as active ingredients , Epilepsy, encephalopathy, and stroke. The method according to claim 1,
The compound is obtained by extracting Curdrania tricuspidata with methanol; Dissolving the methanol extract in water and obtaining a chloroform fraction using a solution-solution dispensing method with chloroform; Wherein the chloroform fraction is separated through silica gel column chromatography to obtain fractions. The method according to claim 1, wherein the fraction is isolated through silica gel column chromatography. ≪ RTI ID = 0.0 > and / or < / RTI > delete The method according to claim 1,
Wherein the inflammation-inhibiting pathway of the Cudraflavanone D or Steppogenin compound is characterized by inhibiting the p-JNK or p-38 pathway, wherein the pathogen is selected from the group consisting of Lou Gehrig's disease, Parkinson's disease, Huntington's disease, Alzheimer's disease , Multiple sclerosis, multiple nervous atrophy, epilepsy, encephalopathy, and stroke. Parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis, multiple nervous atrophy (Cushing's syndrome), including Cudraflavanone D or Steppogenin compounds isolated from Curdrania tricuspidata as active ingredients , Epilepsy, encephalopathy, and stroke.
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Publication number Priority date Publication date Assignee Title
KR20190000086A (en) 2017-06-22 2019-01-02 대구가톨릭대학교산학협력단 Composition comprising compounds isolated from Morus alba for preventing or treating of inflammatory disease
KR20200127424A (en) 2019-05-02 2020-11-11 가천대학교 산학협력단 A composition for preventing or treating Advanced Glycation Endproducts related diseases comprising extracts of Cudrania tricuspidata or compounds isolated therefrom
KR20220020476A (en) 2020-08-11 2022-02-21 극동대학교 산학협력단 Composition comprising steppogenin for prevention or treatment of thrombotic disorders
KR102563495B1 (en) * 2022-08-19 2023-08-10 경북대학교 산학협력단 A composition for preventing, alleviating, or treating cancer comprising steppogenin as an active ingredient
WO2024039164A1 (en) * 2022-08-19 2024-02-22 경북대학교 산학협력단 Composition for preventing, ameliorating, or treating cancer comprising steppogenin as active ingredient

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